Engineered uridine phosphorylase variant enzymes

Abstract

The present invention provides engineered uridine phosphorylase (UP) enzymes, polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing UP enzymes are also provided. The present invention further provides compositions comprising the UP enzymes and methods of using the engineered UP enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

We claim: 
     
       1. A polynucleotide sequence encoding at least one engineered uridine phosphorylase comprising a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 2 or a functional fragment thereof, wherein the polypeptide sequence of said engineered uridine phosphorylase comprises at least one substitution at position 80 and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2. 
     
     
       2. A polynucleotide sequence encoding at least one engineered uridine phosphorylase, said polynucleotide sequence comprises at least 90% sequence identity to SEQ ID NO: 1 wherein the polypeptide sequence of said engineered uridine phosphorylase comprises at least one substitution at position 80. 
     
     
       3. A polynucleotide sequence encoding at least one engineered uridine phosphorylase comprising at least 90% sequence identity to SEQ ID NOs: 3, 245, 593, 775 and/or 867, or a functional fragment thereof. 
     
     
       4. The polynucleotide sequence encoding an engineered uridine phosphorylase of  claim 1 , wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 90% sequence identity to SEQ ID NO: 2, and wherein the polypeptide sequence of said engineered uridine phosphorylase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 6, 7, 9, 14, 14/38/40/146/147/179/235/236, 14/38/86/146/147/235/236/240, 14/40, 14/40/86/147/193/236/240, 14/40/136/179/236/240, 14/40/146/235/236, 14/40/147/181/193/235, 14/40/235, 14/86/146, 14/146/147/181/240, 14/146/236/240, 14/147/193/235/236/240, 14/179/181/193/235/240, 14/235/236, 29, 31, 38/40/86/146/147/179/181, 38/40/86/147/236/240, 40, 40/43/86/146/240, 40/43/86/147/235, 40/43/146/147, 40/43/147/179/236/240, 40/43/147/179/240, 40/43/147/236/240, 40/86/146/235/236, 40/86/147/235/236/240, 40/86/179/235/240, 40/86/235/236/240, 40/146/147/240, 40/147/240, 40/235, 40/235/236/240, 40/236, 40/236/240, 42/235/236, 43/86/147/181/240, 43/146/147/235/236/240, 43/146/179/240, 43/147, 43/147/179/181, 47, 47/88, 64, 73, 80, 86, 86/136/146/147/179/181, 86/136/146/147/179/235/236, 86/147/179/181, 86/235, 86/235/236/240, 86/236/240, 86/240, 92, 97, 99, 103, 103/249, 104, 105, 106, 110, 146/147, 146/147/235/236, 146/235/240, 146/236/240, 146/240, 147/179/181, 147/235/240/249, 157, 167, 179, 179/181, 179/181/193/240, 181, 184, 216, 226, 228, 231, 233, 235, 235/236, 236, 236/240, 237, 239, 240, and 245, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2. 
     
     
       5. The polynucleotide sequence encoding an engineered uridine phosphorylase of  claim 1 , wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 90% sequence identity to SEQ ID NO: 4, and wherein said polypeptide sequence of said engineered uridine phosphorylase further comprises at least one substitution or substitution set at one or more positions selected from 3, 3/9/216, 3/9/216/236, 3/9/235/237, 3/31/47/179/181/216, 3/31/47/179/181/237, 3/31/947/179/216, 3/31/47/179/216/237, 3/31/179, 3/31/179/181, 3/31/179/181/237, 3/31/179/216, 3/31/181/216, 3/31/181/237, 3/47/179/181/216, 3/47/179/216/237, 3/47/181, 3/179, 3/179/181, 3/179/181/216, 3/179/181/237, 3/179/216, 3/179/216/237, 3/179/237, 3/181/216, 3/181/216/237, 3/216/236/240, 9/216/236/237, 9/237, 13, 24, 31, 31/47, 31/47/179/181/237, 31/47/179/216, 31/47/181, 31/47/216, 31/179, 31/179/181, 31/179/216, 31/181, 31/181/216, 31/181/216/237, 31/181/237, 31/216, 31/216/237, 31/236/237/240, 31/237, 33, 46, 47, 47/147/181/231, 47/179/181, 47/179/181/216, 47/179/184, 47/179/216, 47/181/216, 47/181/216/237, 47/181/231, 47/216, 52, 63, 67, 83, 87/160, 92, 95, 97, 99, 100, 101, 105, 106, 108, 111, 137, 151, 152, 155, 159, 160, 170, 173, 177, 179, 179/181, 179/181/216, 179/181/216/237, 179/181/231, 179/181/241, 179/216, 179/228/231, 179/237, 181, 181/216, 181/216/237, 181/237, 183, 185, 188, 189, 191, 201, 216, 216/237, 218, 222, 228, 231, 233, 235, 235/237, 236, 236/237/240, 237, 238, 240, 241, and 248, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4. 
     
     
       6. The polynucleotide sequence encoding an engineered uridine phosphorylase of  claim 1 , wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 90% sequence identity to SEQ ID NO: 246, and wherein said polypeptide sequence of said engineered uridine phosphorylase further comprises at least one substitution or substitution set at one or more positions selected from 3/24/33/47/100/183/185, 3/24/33/47/100/216/228/233, 3/24/33/100/183/185/228, 3/24/33/108, 3/24/47/100, 3/24/47/100/108/111/160/185/233/241, 3/24/47/108/160/241, 3/24/47/160/189, 3/24/47/189/228/233, 3/24/47/228, 3/24/47/228/233, 3/24/95/100, 3/24/95/100/160/189/228/241, 3/24/100, 3/24/100/160/218/241, 3/24/111, 3/24/111/183/228/233/241, 3/24/111/228/233, 3/24/183/185/216, 3/24/189/233, 3/33/47/95/100/241, 3/33/47/100/108/189/216/228/233, 3/33/47/100/111/228, 3/33/47/100/111/233/241, 3/33/47/100/216, 3/33/47/108/111/233, 3/33/47/108/189/233/241, 3/33/160/233, 3/47, 3/47/95/100/108/189/233, 3/47/95/100/111/241, 3/47/95/160/189, 3/47/100/108/183/185/189/241, 3/47/100/160/185, 3/47/100/185/189/228, 3/47/108/111, 3/47/183/189/228/233, 3/47/189, 3/47/228/233, 3/95/100/160/228/233, 3/95/100/183/216/228/233, 3/95/100/183/233, 3/95/185/189/216, 3/95/189, 3/95/233, 3/160, 3/183/185/189/228/233, 3/183/189/228/233, 3/185, 3/185/189, 3/189, 24, 24/33/47, 24/33/47/228/241, 24/33/100/108/241, 24/47/95/100, 24/47/95/100/160/228/233/241, 24/47/185/216/218, 24/47/216, 24/95/183, 24/100/160/233, 24/160/183/185, 24/189/228/233, 33/47/95/100/233, 33/47/95/100/233/241, 33/47/160, 33/47/233, 33/100/183/185, 33/100/185/233, 47, 47/100/111/233, 47/100/189, 47/100/189/233, 47/108/160/228/241, 47/111, 47/160/185/189/233, 47/228/233, 95/100/183, 95/100/189, 95/100/228, 95/100/228/233, 95/100/233, 100/160/185, 100/228/233, 108, 108/183/189/233, 108/185/216/228/233, 160/233, 228, and 228/233, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 246. 
     
     
       7. The polynucleotide sequence encoding an engineered uridine phosphorylase of  claim 1 , wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 90% sequence identity to SEQ ID NO: 594, and wherein said polypeptide sequence of said engineered uridine phosphorylase further comprises at least one substitution or substitution set at one or more positions selected from 6, 6/9/29/40/100/121/126/179/181/189/237, 6/9/121/179/181, 6/46/52/63/97/121/126/179, 6/52/180/181, 6/63/126/179/242, 9, 9/40/46/97/100/106/135/179/181/207/231, 9/52/126/189/242, 9/97/100/106/126/180/207/231, 9/181/242, 29, 40, 46, 52, 52/63/126, 52/179/189, 61, 63, 97, 100, 106, 121, 126, 126/180/189, 135, 142, 179, 180, 181, 189, 201, 207, 230, 231, 236, 237, and 242, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 594. 
     
     
       8. The polynucleotide sequence encoding an engineered uridine phosphorylase of  claim 1 , wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 90% sequence identity to SEQ ID NO: 776, and wherein said polypeptide sequence of said engineered uridine phosphorylase further comprises at least one substitution or substitution set at one or more positions selected from 3, 3/8/22/36/181/235/250, 6, 6/45/51/81/126/226/233, 6/45/51/144, 6/45/51/188/189, 6/45/51/189/208, 6/45/51/189/228/236, 6/45/51/208/233, 6/45/149/188/189/208/233/236, 6/51/126/144/208, 6/51/126/189/208/233/236, 6/51/126/189/231/236, 6/51/126/189/233, 6/51/188/189/208/226/228, 6/51/188/189/236, 6/51/189/208/231/233, 6/51/208/226/233, 6/51/208/231, 6/126/188/231/233, 6/144/208, 6/188/189/208/228/233, 8, 8/36/143/147/235, 8/36/181/235, 8/142/147, 8/147/181/250, 8/147/235, 9, 19, 20, 22, 22/147/181/235/250, 24, 36, 36/143/147/181, 36/143/147/235, 40, 41, 43, 45, 45/51, 45/51/126/144/208/226/228, 45/51/144, 45/51/144/208/226/231/233, 45/51/188/189, 45/51/189/233, 45/51/208/226/231, 45/51/208/233, 45/51/226, 45/126/189, 45/126/189/208/226, 45/144/189/228, 45/144/226/231/233, 45/188/189, 45/188/189/208/228, 45/188/189/226/228, 45/188/189/231/233, 45/189, 45/189/208, 46, 51, 51/126/144/208, 51/126/144/226/231/233/236, 51/126/208, 51/144, 51/144/226, 51/188/189/228, 51/189, 51/189/208/226, 51/208, 51/233, 57, 58, 80, 80/135/147, 81, 81/126/144/188/208/228, 86, 103, 126, 126/144/188/189/226, 134, 135, 141, 142, 143, 143/147/235, 144, 144/188/228, 146, 147, 149, 181, 188, 188/189/233, 189, 207, 208, 208/226/233, 208/228, 208/231/233, 226, 228, 230, 231, 232, 233, 235, 236, 240, and 250, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 776. 
     
     
       9. The polynucleotide sequence encoding an engineered uridine phosphorylase of  claim 1 , wherein the polypeptide sequence of the engineered uridine phosphorylase has at least 90% sequence identity to SEQ ID NO: 868, and wherein said polypeptide sequence of said engineered uridine phosphorylase further comprises at least one substitution or substitution set at one or more positions selected from 6, 6/8/9/126/147, 6/9/24/181/189, 6/9/181/189/235, 6/9/208/233/235, 6/24, 6/9/43/46/181/189, 6/9/43/126/147/189, 6/9/46/103/181/208, 6/9/46/147/240, 6/24/103/189, 6/24/126/189, 6/24/147, 6/46/126/147/181/235/240, 6/46/147/189/240, 6/103, 6/103/147/230/233, 6/103/189/235, 6/126/181/189, 6/126/181/189/235, 6/126/233/235, 6/181/230/233/235, 9/43/46/103/189/233/240, 9/46/126/147/181, 9/46/147/233, 24/43/46/147/230/235, 24/46/126, 24/46/147/189, 24/46/208/230/233/235, 24/103/126/147, 24/103/126/147/181/189/208/233, 24/147, 24/147/189/230/233, 24/181/189/230/233/235, 24/189/230, 24/208, 43/46/126/147/189/240, 43/103/189/208/233, 103/126/189/233/235, 103/147/181, 147/181/233, 189, 189/235, and 208/233, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 868. 
     
     
       10. The polynucleotide sequence of  claim 1 , wherein said polynucleotide sequence comprises a polynucleotide sequence set forth in the odd numbered sequences of SEQ ID NOs: 3, 5, 11, 33, 43, 47, and 245-1195. 
     
     
       11. The polynucleotide sequence of  claim 1 , wherein said polynucleotide sequence is operably linked to a control sequence. 
     
     
       12. The polynucleotide sequence of  claim 1 , wherein said polynucleotide sequence is codon optimized. 
     
     
       13. An expression vector comprising at least one polynucleotide sequence of  claim 1 . 
     
     
       14. A host cell comprising at least one expression vector of  claim 13 . 
     
     
       15. A host cell comprising at least one polynucleotide sequence of  claim 1 . 
     
     
       16. A method of producing an engineered uridine phosphorylase in a host cell, comprising culturing the host cell of  claim 15 , under suitable conditions, such that at least one engineered uridine phosphorylase is produced. 
     
     
       17. The method of  claim 16 , further comprising recovering at least one engineered uridine phosphorylase from the culture and/or host cell. 
     
     
       18. The method of  claim 17 , further comprising the step of purifying said at least one engineered uridine phosphorylase.